Oligonucleotides containing 5-fluorouracil

ABSTRACT

A homo-oligonucleotide of between 2 and 26 monomers of 5-fluorouridine 5&#39;-monophosphate or 5-fluorodeoxyuridine 5&#39;-monophosphate covalently linked via 3&#39;- to 5&#39;-phosphodiester linkages, where at the 3&#39;- or 5&#39;- terminus there is covalently linked a molecule selected from the group consisting of cholesterol, ethyl-spaced adamatane, 1,2-di-hexadecylglycerol and poly-L-lysine. These homo-oligonucleotides exhibit antitumor activity.

CROSS REFERENCES RELATED APPLICATIONS

The present application is an original patent application and iscurrently not known to be related to any co-owned and co-pendingapplication.

GOVERNMENT RIGHTS

The present invention was privately funded. The federal government hasno rights in the present invention.

TECHNICAL FIELD

The present invention is generally related to chemotherapeutictreatments and more specifically to methods for increasing the in-vivohalf-life and target specificity of chemotherapeutic drugs such as5-fluorouracil.

BACKGROUND ART

Anti-metabolite nucleosides and nucleoside analogues have foundwidespread use in the treatment of cancer and other human diseases.6mercaptopurine was found by Elion and co-workers to interfere withpurine metabolism and is used in the treatment of leukemia. Othernucleosides or nucleosides analogues that are of use includetrifluorothymidine, arabinosyl cytidine.

5-Fluorouracil (5-FU) has been used continuously since its developmentin 1957 by Duusinski and Heidelberger (U.S. Pat. No. 2,802,005) for thetreatment of solid tumors of the head, neck, breast, and colon. 5-FU wasoriginally designed to work as an inhibitor of thymidylate synthetase(TS). TS is the enzyme which converts deoxyuridine 5'-O-monophosphate(dUMP) to deoxythymidine 5'-O-monophosphate (dTMP). It is believed that5-FU retards tumor expansion by causing thymidine pools to becomedepleted in rapidly proliferating tumor cells.

Protocols for the administration of 5-FU for treatment of human cancerinvolve infusion of the drug for long periods of time. 5-FU is rapidlymetabolized and excreted with a half-life in-vivo of about 18 minutes.While 5-FU is an effective anti-cancer agent when metabolicallyactivated to become an inhibitor of TS its effectiveness is hampered byrapid metabolism and formation of 2-fluoro-β-alanine (FBAL) which isneurotoxic and cardiotoxic. For these reasons researchers and clinicianshave long desired a method of increasing the therapeutic index andtarget specificity), of 5-FU.

A variety of pro-drug forms of 5-FU have been developed to address theissues of cellular uptake, sustained release, organ distribution, andtransdermal or intestinal uptake that are problematic for the nativedrug. One of the most widely studied pro-drug forms of 5-FU is5'-deoxy-5-fluorouridine (DFUR). DFUR is converted to 5-FU by pyrimidinenucleoside phosphorylase but has better cellular uptake properties than5-FU. Like 5-FU, DFUR also releases FBAL as a toxic metabolite. Othernucleoside analogues of FUr include Tegafur[(1-(2-tetrahydrofuryl)-5-fluorouracil], Ftorafur[R,S-1-(tetrahydro-2-furanyl)-5-fluorouracil] and a variety of5-fluorocytidine derivatives.

A variety of polymeric forms of 5-fluorouridine have also been preparedto provide sustained release of 5-FU. 5-Fluorouracil has been preparedas a conjugate of chito-oligosaccharides and also as a conjugate ofpoly(ethylene glycol). These polymeric forms were designed to providemacromolecular drugs with reduced side-effects and strong anti-tumoractivity and showed good biological activity and low toxicity in animalmodels. The chief advantages of FdU_(n) and FrU_(n) compared to otherpolymeric structures are that the oligonucleotide based oligomers arereadily taken up by cells, perhaps through a facilitated mechanism.Also, enzymatic degradation of the oligomeric 5-fluorodeoxyuridineresults directly in release of the fully activated inhibitor ofthymidylate synthase (FdUMP). Oligonucleotide based polymers retain theadvantages that make other polymers useful such as increasedbioavailability. Homo-oligomeric compositions of other nucleosideantimetabolites benefit from positive increase in bioavailability andcellular uptake. Homo-oligomeric anti-metabolite nucleotides aresynthesized and used as a polymeric drug delivery system.

DISCLOSURE OF INVENTION

Homo-oligomeric 5-fluorouridine and 5-fluorodeoxyuridine (FrU_(n) andFdU_(n), n=oligomer length) are synthesized and used as a polymeric drugdelivery system for production of FdUMP, the potent inhibitor ofthymidylate synthetase (TS) and an important target in cancerchemotherapy. Disclosed are methods of both preparing and utilizingthese novel compositions. The oligomeric compounds of the presentinvention efficiently transverse the cellular membranes of a variety ofcell lines and are degraded to an active form (FdUMP in the case ofFdU_(n)). FdU_(n) is a more effective cytotoxic agent in cell culture ona per residue basis than is monomeric 5-fluorodeoxyuridine. Based ondata from other oligonucleotide drugs these oligomeric compounds have asignificantly longer in-vivo residence time compared with monomericcompounds. The advantages of a longer residence time in-vivo, andgreater cytotoxicity per residue of 5-fluorodeoxyuridine, demonstratesthat oligomeric 5-fluorodeoxyuridine is therapeutically useful at lowerdoses than are monomeric 5-fluorouridine, 5-fluorodeoxyuridine, and5-fluorouracil. Thus, delivery of FdUMP as FdU_(n) is more costeffective on a per dose basis and less neurotoxic and cardiotoxic thandelivery in a monomeric form. The reduced neuro- and cardiotoxicity is aconsequence of lower levels of 2-fluoro-β-alanine (FBAL) released fromthe lower effective dosage. FdU_(n) and FrU_(n) are representative of aclass of homo-oligomeric anti-metabolite compounds that includehomo-oligomeric 6-mercaptopurine, homo-oligomeric araC, andhomo-oligomeric 5-trifluoromethyl thymidine and other homo-oligomers ofbioactive nucleosides and nucleoside analogues. In all cases theoligomeric form allows increased bio-availability and improved cellularuptake.

Delivery of FrU_(n) results in degradation by nuclease to FUMP. FUMP isacted upon by kinases to form FUTP. FUTP is incorporated into cellularRNA. Normal turnover at cellular RNA releases FUMP which may beconverted by a series of enzymatic steps to form FdUMP. Delivery ofFrU_(n) thus results in sustained production of FdUMP that may beimportant in inhibiting DNA replication in slowly dividing cells. Thecompounds of the present invention also have an added advantage in beinghomopolymers in synthesis. In a range of lengths (e.g. 8 to 12),acceptable for administration as a chemotherapeutic agent, sucholigomers may be synthesized in solution (and the proper length speciesseparated). This method is much more cost-effective than solid-phasesynthesis and HPLC purification which are necessary in antisenseoligonucleotides. Thus the present invention discloses a method ofdelivering anti-cancer and anti-viral nucleosides in oligomeric form toovercome the rapid metabolism and excretion of monomeric nucleosides,nucleoside bases, and nucleoside analogues.

OBJECTS OF THE INVENTION

Delivery of these nucleosides as homo-oligomeric nucleotides confersseveral distinct advantages relative to their delivery as nucleosidebases, nucleosides, or analogues:

The polymeric form increases bioavailability relative to the monomericforms. The increased bioavailability results in a more convenientadministration schedule than available for the monomeric forms.

The oligonucleotide compounds are actively taken up by cells resultingin a higher intracellular concentration from the oligonucleotide baseddrug than for the monomeric forms. This results in a greater proportionof drug inside tumor cells.

Enzymatic degradation of these homo-oligomeric nucleotides by3'-exonucleases releases the nucleoside 5'-O-monophosphate. Thus, theNMPs are the fully metabolically activated product, e.g. FdUMP, isreadily converted to the fully activated metabolic product, e.g.,araCTP. Several steps of metabolic activation may be bypassed andnonproductive pathways of metabolic activation are avoided.

The before mentioned advantages significantly reduce the dose requiredfor a positive biological response and reduce dose-dependent toxicside-effects. Therefore, these homo-oligomeric nucleotides providehigher therapeutic indices while additionally being more cost-effectivethan their monomeric counterparts.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the chemical structure of a monomer ofoligomeric 5-fluorouridine-5'-O-monophosphate;

FIG. 2 is a diagrammatic illustration of the metabolic pathway of 5-FUand the entry points for the products of exonuclease action on FdU_(n)and FrU_(n) ;

FIG. 3 is a diagrammatic illustration showing a preferred method ofpreparing homo-oligomeric 5-fluorodeoxyuridine-5'-O-monophosphate;

FIG. 4 is a diagrammatic illustration showing a preferred method ofpreparing homo-oligomeric 5-fluorouridine-5'-O-monophosphate;

FIG. 5 is a diagrammatic illustration showing a preferred method ofpreparing [4-¹⁴ C]5-fluorouridine for incorporation into oligomers;

FIG. 6 is a diagrammatic illustration showing a preferred method ofpreparation of the reactive phosphoramidites of cholesterol, anethyl-spaced adamantane, and 1,2-di-hexadecyl glycerol at the 5'termini; and

FIG. 7 is a diagrammatic illustration showing a preferred method ofpreparation of the reactive phosphoramidites of cholesterol, anethyl-spaced adamantane, and 1,2-di-hexadecyl glycerol at the 3'termini.

MODES FOR CARRYING OUT THE INVENTION

I. Introduction

The delivery of nucleosides and nucleoside analogues that haveanti-cancer and anti-vital activity as monomers in the form ofhomo-oligomeric nucleotides (oligonucleotides composed of a single typeof nucleotide, e.g., dA) has significant advantages relative to theirdelivery as monomers.

It should be apparent to individuals skilled in the art that equivalencesubstitutions for 5-FU may be made, for example (1) pyrimidine analoguessuch as CYTARABINE™"ara-C", azauridine, azaribine, 5-chlorodeoxyuridine,5-bromodeoxyuridine, 5-iododeoxyuridine, 5-trifioromethyldeoxyuridine;and (2) purine analogues such as 6-thioguanine, 6-mercaptapurine,azathioprine, "2'deoxycofermycin" PENTOSTATIN™,erythrohydroxynonyladenine.

These agents are or have been employed as anti-cancer immunosuppressanttherapeutics. Since they generally have short half-lives and theirutility is limited by the toxicity of their metabolites they may allshow significant advantages as homo-oligomeric polymers relative totheir monomeric congeners.

For example, homo-oligomeric nucleotides, (due to their higher molecularweight), have increased bioavailability relative to their constituentmononucleosides (which are readily metabolized and excreted).Homo-oligomeric nucleotides may also be taken up via a receptor and moreefficiently gain entry to the cell than mononucleotides. Once taken upby cells, such compounds are readily degraded by exonucleases to their5'-O-monophosphate form (frequently the bio-active form of thenucleoside).

The present invention describes a method of utilizing oligomeric formsof 5-FU (e.g., FdU_(n) FrU_(n) [FIG. 1]) for anti-cancer treatments.5-Fluorouracil (5-FU) is used frequently in the treatment of solidtumors of the gastrointestinal tract, breast, ovary, and skin. In aneffort to increase clinical effectiveness, numerous studies have beenconducted to better understand the mechanisms by which 5-FU iscytotoxic. Despite the enormity of these efforts the objective responserates for 5-FU chemotherapy, either singularly or in combination withother drugs, range from 10 to 33%, with cures being achieved rarely.

The present invention teaches novel compositions and methods of: (1)delivering 5-FU and related compounds in order to maximize intracellularconcentration of the metabolite(s) responsible for 5-FU cancer cellcytotoxicity; (2) overcoming 5-FU's relatively poor bioavailability andthe toxicity of its metabolites (chiefly the neurotoxicity of FBAL); and(3) enhancing the delivery of cytotoxic metabolite(s) such that (a) thecytotoxic metabolite delivery is timed for optimum tumor celldestruction, (b) the cytoxic metabolites are tumor target specific, and(c) tumor cell uptake is optimized.

II. Synthesis of FdU_(n) and FrU_(n) Species

All synthetic reactions are performed under an inert atmosphere inoven-dried glassware and followed by thin layer chromatography inappropriate solvent systems. Intermediates are preferably analyzed by300 MHz 1H NMR spectroscopy and critical intermediates and finalproducts are preferably characterized by 1H, 13C NMR spectroscopy andmass spectrometry. Solvents and starting reagents may be purchased fromAldrich Chemical Company. Radioactive substances may be purchased fromDupont New England Nuclear.

Those skilled in the art of biochemical synthesis may produce FdU_(n)and FrU_(n) and their conjugates, according to the present invention,wherein the following syntheses are currently preferred. Otherhomo-oligomeric nucleotides may be prepared in completely analogousfashion.

5'-O-[4,4'-dimethoxytrityl]-5-fluorouridine

5.244 gins. (20 mMoles) of 5-fluorouridine is dissolved in 20 mLanhydrous pyridine and then dried under vacuum. This procedure isrepeated at least one more time in order to make 5-fluorouridine devoidof moisture. The dried 5-fluorouridine is then dissolved in 20 mL ofpyridine. 8.131 gms (24 mMoles, 1.2 eq.) of 4,4'-dimethoxy tritylchloride is added as solid in 4 fractions over a period of 2 hours. Thereaction mixture is then stirred at room temperature overnight and isthen diluted with 25 mL of dichloromethane and washed with saturatedsodium bicarbonate solution. The aqueous layer is then washed with 20mLdichloromethane. The organic layers are mixed and washed with saturatedbicarbonate solution two more times. The organic layer is then driedover anhydrous sodium sulphate. The solvent is removed under vacuum toyield a yellow foam which is purified on a silica gel column using 90:10chloroform:methanol as eluent. The 4, 4'-dimethoxytrityl derivative of5-fluorodeoxyuridine is prepared in an analogous manner.

Rf=0.523 in chloroform:methanol(90:10)

MS, m/z 303.1 (DMtrityl cation), 587.1 (M+Na⁺), 609.1 (M+2Na⁺)

5'-O-[4,4'-dimethoxytrityl]-[2'-O-t-butyl-dimethylsilyl]-5-fluorouridine

10 gms(18 mMoles) of 5'-O-[4,4'-dimethoxytrityl]-5-fluorouridine isdried from pyridine under vacuum to make it free of moisture and thendissolved in 40mL pyridine and 4.95 imidazole g (72 mMoles, 4 eq) isadded. The solution is stirred for an hour and then 3 gms (20 mMoles,1.1 eq) tert-butyl dimethyl silyl chloride is added as solid in a singleportion. The reaction is allowed to proceed for 5 hours at roomtemperature. A similar work up with aqueous saturated sodium bicarbonateis followed. The crude product is purified on a silica gel column using65:35 hexane:ethyl acetate as eluent. The bis-silyl and two monosilylproducts are separated and the correct monosilyl isomer is used forphosphorylation reaction.

Rf=0.457 (bis silyl product) 0.286 (2'-silyl isomer) 0.114 (3'-silylisomer)in 65:35 hexane:ethyl acetate

MS, m/z 303.2 (Dmtrityl cation), 815.5 (M+Na⁺) - bis silyl product m/z303.0 (DMtrityl cation), 701.0 (M+Na⁺) - 2'-silyl isomer m/z 303.0(DMtrityl cation), 701.0 (M+Na⁺) - 3'-silyl isomer

5'-O-[4,4'-dimethoxytrityl]-[3'-O-diisoprop ylcyanoethylphosphonamidic-chloride]-[2'-O-t-butyl-dimethylsilyl]-5-fluorouridine

To a stirred THF solution of 1.5 mL(11.6 mMoles, 4 eq.)diisopropylethylamine, 88 mg. (0.73 mMoles, 0.25 eq.) DMAP, and 1.03mL(4.35 mMoles, 1.5 eq. ) of2-cyanoethyl-N,N-diisopropylchlorophosphoramidite are added, dropwise, asolution of 2 g (2.9 mMoles) of 2'-monosilyl trityl 5-FU in equal volumeof THF at room temperature with stirring. After stirring for 4 hours thereaction mixture is worked up by diluting with 20 mL ethyl acetate andwashed five times with saturated sodium chloride solution. The solventis removed under vacuum and the residue is purified on a silica gelcolumn using 90:10 chloroform:triethylamine as eluent. The productobtained is a white foam. The phosphoramidite of the5'-O-[4-4"-dimethyloxytrityl]-5-fluorodeoxyuridine is prepared in a likemanner.

Rf=0.45 in 90:10 chloroform:triethylamine

MS, m/z 303.1 (trityl cation), 901.3 (M+Na⁺)

A. Synthesis of [4-¹⁴ C] 5-fluorouridine

The following methodology may be useful to those practicing theinvention wherein radio-labelled forms of the oligomers of the presentinvention are desired. The synthesis of [4-¹⁴ C] 5-fluorouridine isshown in FIG. 5. The ¹⁴ C isotope is preferred over the more readilyobtained ³ H isotope since it is not exchanged in acidic subcellularcompartments or tissues. Although [2-¹⁴ C] 5-fluorouridine is known inthe art, the [4-¹⁴ C] compound is not. Introduction of ⁴ C at the 4position is preferred following metabolism since it is retained in FBAL(a neurotoxic catabolite of 5-fluorouridine). The ¹⁴ C probe allowsstraightforward analysis of metabolism by radio-detected HPLC. Thesynthetic procedure for preparation of [4-¹⁴ C]-5-fluorouridine (FIG. 5)is based on the synthesis of 2', 3', 5'-tri-O-benzoyl[4-³ C] uridine(Roberts & Poulter). The original source of ¹⁴ C for this synthesis is1-¹⁴ C sodium acetate. The acetate is brominated at the 2 position andthe bromine is then displaced by cyanide. The cyano group is thenreduced over a platinum catalyst and the terminal amine reacted with theisothiocyanate to form the dihydropyrimidine. The 5,6 double bond isintroduced by dehydrobromination to form uracil. Glycosylation isaccomplished by treating the O-acetyl sugar with a Lewis acid inacetonitrile after protecting the uracil carbonyls as theO-trimethylsilyl derivatives. Fluorination at the desired position isthen induced by the Robins method (treatment with trifluoromethylfluoride). Formation of the deoxynucleoside is accomplished by selectivereduction of the 2'-OH using tri-butyl tin hydride after protection ofthe 3' and 5' hydroxyls with di-isopropyl disiloxane in anhydrous THF.The 5'-O-4,4'-dimethoxytrityl derivatives of [4-¹⁴ C]5-fluorouridine and[4-¹⁴ C]-5-fluorodeoxyuridine are prepared by treating the nucleosideswith 4,4'-dimethoxytrityl chloride in pyridine. The2'-O-t-butyl-dimethyl-silyl derivative of the ribonucleoside is preparedby treatment with t-butyl-dimethyl-silyl-chloride in dry THF and isseparated from the 3'-isomer and the bis-silylated product bypreparative normal phase HPLC. Reactive phosphoramidites are prepared byreaction with cyanoethylphosphonamidic-chloride using Hunigs base in dryTHF and purified by column chromatography on silica gel.

B. Synthesis and Purification of FdU_(n) and FrU_(n)

The controlled pore glass support used for solid phase synthesis ofFdU_(n) is prepared by reacting the5'-O-[4,4'-dimethoxytrityl]-5-fluorodeoxyuridine first with succinicanhydride to form the 3'-O-succinate and then with long-chain alkylaminecontrolled pore glass in the presence of catalytic amounts ofdimethylaminopyridine and stochiometric amounts of1-(3-dimethylaminopropyl)-ethylcarbodiimide in anhydrous pyridine. Thecontrolled pore glass support used for solid phase synthesis of FrU_(n)is prepared by reacting5'-O-[4,4'-dimethoxytrityl]-[2'-O-t-butyl-dimethylsilyl]-5-fluorouridinein an analogous manner. The loading capacity for both FdU_(n) andFrU_(n) derivatized CPG is determined by trityl analysis at 600 nmfollowing exposure to acid. The derivatized support is then suspended ina mixture of acetic anhydride (5 mL), 2,4,6 collidine (7 mL), and DMAP(3 g) in anhydrous THF (100 mL) and shaken for two hours. The CPG isthen washed with dichloromethane and loaded at the appropriate capacity,e.g., 1 or 10 μmole, into columns for use in an ABI solid-phase DNAsynthesizer.

FdU_(n) and FrU_(n) synthesis is preferably performed on an AppliedBiosystems 380B DNA synthesizer. The synthesis conditions areessentially the same as the standard 10 μmole cycle, but utilizes the5'-O-[4,4'-dimethoxytrityl]-[3'-O-diisopropylcyanoethyl-phosphonamidic-chloride]-[2'-O-t-butyl-dimethylsilyl]-5-fluorouridinefor FrU_(n) preparation, (or5'-O-[4,4'-dimethoxytrityl]-[3'-O-diisopropylcyanoethyl-phosphonamidic-chloride]-5-fluorodeoxyuridinefor FdU_(n) preparation) as the sole nucleoside phosphoramidites in eachcoupling cycle. The number of coupling cycles may be varied from two toany number with the most useful number of cycles being eight to 24.After synthesis, the FdU_(n) is cleaved from the CPG support bytreatment with 28% ammonium hydroxide (90 min, room temperature). ForFrU_(n), the ammonium solution is then heated in a sealed tube at 55° C.overnight to remove the protecting groups. The crude material is thendesalted on Sephadex G-25 prior to purification by HPLC. The solution isdecanted off and the CPG is washed with anhydrous methanol. Thecompletely deprotected material is then purified by HPLC. Aliquots of200-250 ODU are purified on a Protein-Pak DEAE-5PW anion exchange column(22.5 mmx 150 mm). A gradient from 0-0.15M sodium perchlorate (90 min)at 5 ml/min is used to elute the product. Pure fractions are combined,lyophilized, and desalted on Sephadex G-25. All FdU_(n) and FrU_(n) ishandled with gloves and sterile water and consumables are used at alltimes. (FIGS. 3 & 4).

III. Enhanced Methods of Delivery

A. Timing Production of Cytotoxic Metabolite(s) to Cell CycleSusceptibility

Mammalian cells internalize oligonucleotides and their derivatives by anendocytotic mechanism (see review Vlassov and Yakubov, 1991 and Crooke,1993). It is clear that oligonucleotide derivatives can affect functionsof specific nucleic acids in vitro and inside cells (Degols et al.,1991; Helene and Toulme, 1990; Cohen, 1989). Incubation of cells withradio-labeled oligonucleotides results in binding of the radioactivematerial to the cells and its accumulation in the cell nuclei (Vlassovet al., 1986 and Zamecnick et al., 1986). Uptake is relatively rapid ascells exposed to a 20-mer at a concentration of 20 μM produced acellular concentration of 1.5 μM within 15 minutes (Goodchild et al.,1988). Autoradiography of cells reveals label in both cytoplasm andnucleus of the cells (Iversen et al., 1993). Further, oligonucleotideswith an alkylating group, an aromatic nitrogen mustard at the 5'terminal phosphate provide direct evidence of the intracellularlocalization (Karpova et al., 1980 and Vlassov et al., 1986b).

Detailed studies on the mechanism of cell uptake have been performedwith fluorescently labelled oligonucleotides. Oligonucleotides are takenup by cells in a saturable manner compatible with endocytosis. Maximalbinding is achieved within 2 hours of incubation. The process is slowedby decreased temperature and inhibitors of endocytosis such asdeoxyglucose, cytochalasin B and sodium azide. Further 20% of thematerial was taken into nuclei and 50% was associated with mitochondria,lysosomes and other vesicular structures (Loke et al., 1989; Neckers,1989; and Iversen et al., 1993). Once in the cytoplasm but not invessicles the oligonucleotide is rapidly transported to the nucleus(Chin et al., 1990 and Leonetti et al., 1991). Different cell lines binddifferent amounts of oligonucleotides. The efficiency of oligonucleotideinternalization depends on the conditions of cell growth. An increase inthe cell monolayer density from 8×104 to 5×10⁵ cells/cm² resulted in a3-fold decrease in maximal binding of the oligomer per cell (Ceruzzi andDraper, 1989).

Since oligonucleotide binding is saturable, a receptor at the cellsurface capable of mediating cellular uptake is suggested. The bindingis trypsin sensitive indicating the transport involves a protein (Emlenet al., 1988). An oligonucleotide with an N-hydroxy-succinimidyl (NHS)ester attached to the "Denny-Jaffe" cross-linking reagent and anoligonucleotide with an aminolinker group at the 5' end is incubated incells in the dark. Then cells are exposed to ultraviolet light whichactivates the cross-linking reagent to associated proteins revealing an80-kDa protein, the putative receptor (Neckers, 1993 and Geselowitz andNeckers, 1992). DNA binding is most likely mediated by this receptor inplatelets, leukocytes and lymphocytes (Dorsch, 1981; Ohlbaum et al.,1979; Bennett et al., 1985; and Diamantstein and Blitstein-Willinger1978). Finally, this receptor is linked to cellular functions andpathophysiology (Bennett et al., 1987 and Bennett et al., 1986).

Tumor cells are for the most part rapidly dividing. Many anti-tumordrugs, including 5-FU and its derivatives, are designed to inhibit DNAsynthesis. FdUMP inhibits thymidylate synthase (TS) and results in alack of dTTP which is one of the four nucleotides necessary for DNAreplication and hence cell-division. The thymidineless state has notbeen definitively shown to be cytotoxic apart from its inhibition of DNAreplication and cell division. Cells undergo DNA replication duringS-phase of the cell cycle. Delivery of FdU_(n) to cells at the onset ofS-phase is thought to efficiently inhibit dTTP production and shutdownDNA replication and cell division. Cells prepared to undergoDNA-replication and inhibited from doing so then presumablyself-destruct (or are exceedingly vulnerable to external cytotoxicagents such as free radicals). Some tumor cells may be dividing onlyslowly and delivery of FdU_(n) at an inopportune point in the cell cyclemay not have the desired cytotoxic effect. Whatever TS is expressed isinhibited but the FdUMP produced eventually degrades with little impacton the ability of the cell to replicate DNA or undergo cell division.Slowly dividing cells require sustained release of FdUMP so that it ispresent in sufficient quantities whenever DNA synthesis proceeds.Conjugation of FdU_(n) at the 3' or 5' terminal hydroxyl with a bulkysubstituent may slow exonuclease action and result in steady statelevels of FdUMP that are inhibitory of DNA replication over a prolongedtime. Alternatively, delivery of FrU_(n) results in FUMP being releasedby exonuclease action. FUMP is readily metabolized to FUTP andincorporated into all species of RNA. Natural degradation of RNAreleases FUMP that may be converted to FUDP and reduced to FdUDP byribonucleotide reductase and converted to FdUMP. This process results insteady-state levels of FdUMP that inhibit DNA replication and celldivision. Delivery of FdU_(n) and FrU_(n) simultaneously is also aneffective way in which to prevent tumor cells from undergoing DNAreplication and cell division whether they are just entering S-phase andbeginning DNA replication or are in other phases of the cell cycle whereimmediate high levels of FdUMP have little effect.

B. Methods of Producing Target Specific Delivery

Transformed cells that constitute a solid tumor are known to expresscharacteristic antigenic components on their cellular surface. Thisobservation is the basis for radio-immunoconjuage therapy in whichradio-labelled monoclonal antibodies specific for tumor cell antigensare used to deliver a toxic dose of radioactivity directly to tumorcells. Similarly, conjugation of homo-oligomeric FdUMP via a labilelinker with an antibody fragment directs the homo-oligomeric FdUMP totumor cells. Cleavage of the labile linker releases homo-oligomericFdUMP in high concentrations in the vicinity of the tumor and in lowconcentrations elsewhere. This improves the therapeutic index for thedrug by lowering the dose needed to be cytotoxic to tumors because it ispresent in higher concentrations locally near the tumor. Release ofcytotoxic metabolites (FBAL) is thus decreased.

C. Methods of Increasing Cellular Uptake

The delivery of 5-fluorodeoxyuridine 5'-O monophosphate (FdUMP) ashomo-oligomeric 5-fluorouridine leads to increased cellular uptake.Other homo-oligomeric anti-metabolites nucleotides also have increasedcellular uptake. It is known that the nucleoside base 5-fluoruracil(5-FU) easily penetrates the cellular membrane and that the nucleosides5-fluorodeoxyuridine (FdU) and 5-fluorouridine (FrU) also gain entry tothe cell. The mononucleotide FdUMP, the species responsible forthymidylate synthase (TS) inhibition, does not appreciably penetrate thecellular membrane. Data with FdU_(n) with both phosphorothioate andphosphodiester backbones strongly suggests that these homo-oligomersefficiently gain entry to the cell. The efficiency of cellular uptake isunderscored by the increased effectiveness as cytotoxic agents ofFdU_(n) per residue of FdU relative to monomeric FdU_(n). The increasedcellular uptake is a function of the oligomeric nature of thesecompounds. Entry of one FdU_(n) releases n residues of FdUMP. Theincreased effectiveness of homo-oligomeric FdUMP relative to monomericnucleosides or nucleoside bases is also a function of the naturaldegradation processes in the cell that degrade the oligonucleotide via3' exonuclease action to release FdUMP (the potent inhibitor of TS).Direct production of FdUMP circumvents the steps of metabolism requiredfor production of FdUMP from 5-FU, FrU, or FdU. FdUMP in monomeric formdoes not appreciably penetrate the cellular membrane. This is also animportant distinction between homo-oligomeric FdUMP and oligomers orpolymers that are based on ethylene glycol or oligosaccharide polymericbackbones. Such organic based polymers do not penetrate the cellularmembrane intact because they do not interact with the cellular membranein the same way as do oligonucleotides with either a phosphodiester orphosphorothioate backbone. Further, even if they were taken up intact bya cell, their chemical or enzymatic degradation would not directly yieldFdUMP, but a precursor to FdUMP that must be enzymatically activated.

Cellular uptake of FdU_(n) and FrU_(n) is enhanced by conjugation ateither the 3' or the 5' terminal hydroxyl with lipophilic moities suchas cholesterol or cationic amino acids such as L-lysine orpoly-L-lysine. Such derivitization has proven useful in studies withother oligonucleotides in improving cellular uptake above the relativelygood uptake of the native oligonucleotides. Such conjugation increasesthe affinity of the oligonucleotides for the cellular membrane orreduces electrostatic repulsions for the oligonucleotide while in thehydrophobic membrane or possibly promotes cellular uptake via receptorsthat recognize the conjugated species.

The presence of lipophilic or cationic moieties at the termini effectssubcellular distribution and metabolism. In particular, 3'-terminusderivatization hinders 3'-exonuclease activity and retards the enzymaticdegradation of FdU_(n) and FrU_(n). Preferably a preparation of FdU_(n)or FrU_(n) with cholesterol, ethyl-spaced adamantane, and1,2-di-hexadecyl glycerol at either the 5' or the 3' termini areutilized.

It is preferred that a preparation of 5'-derivatized FdU_(n) or FrU_(n)is made by reaction of the 5'-hydroxyl of the oligomeric compound whilestill attached to controlled pore glass beads with a reactivehydrophobic or lipophilic phosphoramidite. The preparation of thereactive phosphoramidites of cholesterol, an ethyl-spaced adamantane,and 1,2-di-hexadecyl glycerol is shown in FIG. 7.

The dry alcohols are dissolved in anhydrous dichloromethane and reactedwith 2-cyanoethyl-N,N-diisopropylphosphoramidochloridite by usingdiisopropylethylamine as a base. The reaction proceeds under an Argonatmosphere and is quenched with ethyl acetate and purified by flashchromatography on silica gel. The phosphoramidites are then used in anABI solid-phase DNA synthesizer. Preferably a normal coupling cycle isutilized (this may be modified so as to improve coupling efficiency).The oligonucleotides are then removed from the CPG support and purifiedas described herein before.

The attachment of lipophilic moieties to the 3' terminus may beaccomplished as depicted in FIG. 7. The same alcohols shown in FIG. 6,cholesterol, ethyl-spaced adamantane, and 1,2-di-hexadecyl glycerol arefirst tosylated. The tosyl group is then displaced by the sodium salt ofsolketal. The acetonide is then deprotected with mild acid. The primaryalcohol is then dimethoxytritylated and the secondary alcohol reactedwith succinic anhydride to form the succinates. These are then reactedwith long-chain alkylamine controlled pore glass in the presence ofcatalytic amounts of dimethylaminopyridine and stochiometric amounts of1 -(3-dimethylaminopropyl)-ethylcarbodiimide in anhydrous pyridine. Theloading capacity of the derivatized CPG is determined by trityl analysisat 600 nm following exposure to acid. The derivatized support is thensuspended in a mixture of acetic anhydride (5 mL), 2,4,6 collidine (7mL), and DMAP (3 g) in anhydrous THF (100 mL) and shaken for two hours.The CPG is then washed with dichloromethane and loaded at theappropriate capacity, e.g., 1 or 10 μmole, into columns for use in anABI solid-phase DNA synthesizer.

IV. Biological Evaluation of FdU_(n) and FrU_(n) and their Conjugates

A variety of pro-drug forms of 5-FU have been developed to address theissues of cellular uptake, sustained release, organ distribution, andtransdermal or intestinal uptake that are problematic for the nativedrug. One of the most widely studied pro-drug forms of 5-FU is5'-deoxy-5-fluorouridine (DFUR). DFUR is converted to 5-FU by pyrimidinenucleoside phosphorylase but has better cellular uptake properties than5-FU. Like 5-FU, DFUR also releases FBAL as a toxic metabolite. Othernucleoside analogues of FUr include Tegafur[(1-(2-tetrahydrofuryl)-5-fluoruracil], Ftorafur [R,S-1-(tetrahydro-2-furanyl)-5-fluorouracil] and a variety of5-fluorocytidine derivatives.

A variety of polymeric forms of 5-fluorouridine have also been preparedto provide sustained release of 5-FU. 5-Fluorouracil has been preparedas both conjugates of chito-oligosaccharides and poly(ethylene glycol).These polymeric forms were designed to provide macromolecular drugs withreduced side-effects and strong anti-tumor activity and showed goodbiological activity and low toxicity in animal models. The chiefadvantages of FdU_(n) and FrU_(n) compared to other polymeric structuresare that the oligonucleotide based oligomers are readily taken up bycells (most likely through a facilitated mechanism ). Also, enzymaticdegradation of the oligomeric 5-fluorodeoxyuridine results directly inrelease of the fully activated inhibitor of thymidylate synthase(FdUMP). Oligonucleotide based polymers retain the advantages that makeother polymers useful (e.g., increased bioavailability).

Data demonstrates that oligomeric 5-fluorodeoxyuridine is more potentthan monomeric 5-fluorouridine. Rat hepatocytes, H4llE cells, wereplated at a density of 1×10⁴ cells per well in a 96 well microtiterplate and allowed 18 hours to become adherent. The cells were bathed inRPMI 1640 culture medium with 10% fetal calf serum containing FdU_(n)compounds or monomeric 5-fluorodeoxyuridine at concentrations of 0,0.01, 0.03, 0.1, 0.3, 1.0, 3.0, and 10 μM. After 48 h, MTT(3-(4,5-dimethylthizoyl-2-yl)-2,5-diphenyl tetrazolium bromide) wasadded and the resulting insoluble formazon production was monitoredspectrophotometrically at 540 nm. Cell viability was calculated from theamount of formazon produced. Studies with oligomers of length 8, 12, and16 (with both normal phosphodiester and thiophosphate backbones) havedemonstrated the following:

                  TABLE I                                                         ______________________________________                                        Effects of length and backbone on cytoxicity                                  of oligomeric 5-fluorouridine                                                 Length P(O) LD.sub.50                                                                           Relative  P(S) LD.sub.50                                                                         Relative                                 n =    FdU/FdU-N.sup.1                                                                          Potency.sup.2                                                                           FdU/FdU-N                                                                              Potency                                  ______________________________________                                         8     14.7       1.8       1.8      0.22                                     12     28.9       2.4       2.7      0.22                                     16     51.6       3.2       3.4      0.21                                     ______________________________________                                         .sup.1 The ratio of the estimated lethal dose to 50 percent of the cells      in culture for fluorouridine monomer over fluorouridine polymer of length     N, are defined in the left column. P(O) = phosphodiester backbone and P(S     = phosphorothioate backbone.                                                  .sup.2 The relative potency is established as the ratio of LD.sub.50 s        divided by the number of fluorouridine residues to the polymer. This          provides the potency per residue of fluorouridine for direct comparison. 

¹ The ratio of the estimated lethal dose to 50 percent of the cells inculture for fluorouridine monomer over fluorouridine polymer of lengthN, are defined in the left column. P(O)=phosphodiester backbone andP(S)= phosphorothioate backbone.

² The relative potency is established as the ratio of LD₅₀ s divided bythe number of fluorouridine residues to the polymer. This provides thepotency per residue of fluorouridine for direct comparison. Thus:

the cytotoxic potency produced by oligomeric 5-fluorouridine is greaterper residue than that produced by monomeric 5-fluorouridine;

the hydrolysis of the backbone to liberate FdUMP is essential as thenuclease resistant phosphorothioate backbone demonstrates diminishedpotency relative to the phosphodiester; and

increasing the length of the phosphodiester homo-oligomeric nucleotideproduces increased potency per residue of 5-fluorouridine.

The data suggest oligonucleotide transport across cell membranes, and/orcircumvention of enzymatic steps required for conversion to FdUMP,increase the potency of the oligomeric compounds relative to monomericnucleosides.

The present inventors have also studied the cellular binding andtransport of oligomeric 5-fluorouridine across cell membranes. Thesestudies provide evidence of a cellular transport system foroligonucleotides that does not transport mononucleotides. Nekkers hasshown that antisense oligonucleotides are taken up intact by cells in alength-dependent and charge-dependent manner. Mutant cell (which havelost some features of oligonucleotide transport) lines were derived fromthe rat liver hepatocyte, H4lle cells, and are referred to as AMRC1.7and AMRC1.12 cells. Preliminary data concerning these cells and thecomparison of FdU and FdU₈ is shown in Table II. The data indicate thatthere is a cell-dependent uptake (or processing) phenomena that effectsthe relative potency of the oligomeric 5-fluorouridine. The oligomericcompound is more potent on a per residue basis than the mononucleosidein all cell lines except those deficient in oligonucleotide uptake. Wehave evaluated the cytotoxicity of dU and dU₈ using these same cells andassay methods and observed no toxicity up to 10 uM concentrations. Thus,contaminants from synthesis that might persist following purificationare not potential explanations for the cytotoxicity observations. Thesedata clearly demonstrate the differences between cell lines and thecytotoxic response to oligomeric 5-fluorodeoxyuridine. This provided therationale for our evaluating multiple cell types for their response to avariety of oligomeric 5-fluorouridine compounds:

                  TABLE II                                                        ______________________________________                                        Comparison of FdU-8 with FdU cytotoxicity                                     in reduction of a variety of cell lines                                       Cell Type      LD.sub.50 FdU/FdU-8                                                                        Relative Potency                                  ______________________________________                                        H4IIe - Rat Hepatocyte                                                                       35.6         4.20                                              AMRC1.7        7.1          0.89                                              AMRC1.12       5.8          0.73                                              3T3 Mouse Fibroblast                                                                         9.8          1.23                                              168 Metastatic mouse cell                                                                    9.6          1.2                                               410 Highly Metastatic                                                                        10.7         1.34                                              mouse cell                                                                    ______________________________________                                    

Studies concerning the pharmacokinetics of oligonucleotides have alsobeen conducted. Work has been completed in the mouse, rat, rabbit,monkey, and human and has involved a number of differentoligonucleotides. While the pharmacokinetic behavior of anoligonucleotide cannot be predicted at this time, we have observedelimination half-life ranging from 7 to 57 hours for phosphorothioateoligonucleotides intravenously injected into mice. Hence, it seemsreasonable to assume the pharmacokinetic behavior of oligomeric5-fluorouridine will most likely involve a half-life of several hours.Since the half life of 5-FU is about 15 minutes in humans, theoligomeric 5-fluorouridine compounds are very likely to have asignificantly longer residence time. This should result in a substantialreduction in the dose required to produce an equivalent plasmaconcentration. The reduction in total body burden combined withincreased potency of 5-fluorouridine per residue indicates a potentialimprovement in treatment of cancers with reduced side effects. Thissuggests a substantial improvement in the therapeutic index of FdUanti-metabolite in the clinical management of cancer. Delivery of5-fluorouridine in oligomeric form should result in drugs that are moreselective, more powerful, and more bioavailable compared to theirmonomeric counterparts. Further, the advances made in combinationchemotherapy involving 5-FU will still be useful with these oligomericforms. In particular, leucovorin (folinic acid) is expected topotentiate FdU_(n) by increasing 5, 10-CH₂ -tetrahydrofolate levels.This will result in a greater preponderance of stable ternary complexesinvolving FdUMP derived from FdU_(n), TS and 5,10-CH₂ FAH₄.

The methods described herein for 5-FU delivery as an anti-cancer agentmay be generalized to other nucleoside analogues that are anti-cancer oranti-viral agents. Schmidt et al. have proposed incorporation of5-fluoro-2'-deoxycytidine into oligodeoxynucleotides. They proposed thatsuch selectively incorporated fluoropyrimidines could be used to studythe mechanism of cytosine methyl transferases. However, the presentspecification is the first proposed synthesis of oligomeric compoundsconsisting solely of nucleosides that have anti-cancer activity oranti-viral activity as monomers.

A. Methods of Pharmacokinetic and Toxicological Analysis

Prior to undertaking animal studies on the efficacy of oligomeric5-fluorouridine compounds for tumor reduction, a toxicology study usingSprague-Dawley rats is preferably conducted. (These animals arepreferred in order that sufficient blood and urine samples may becollected for analysis). Three groups with five males and five femalesin each group are preferably studied. Group one receives a salinesolution. Group two receives the functionalized oligomeric5-fluorouridine compound with the apparent most promising cell-cultureperformance. Group two is sacrificed on day 8. Group three is treated asis group 2 but is sacrificed on day 28. The oligomeric 5-fluorouridinecompounds are delivered at a dose of 1000 mg/m² by using a 7 day Alzetinfusion pump implanted subcutaneously. Each rat is identified by aunique number on the tail with the numbers posted on the cage. Rats arefed a diet of Purina rodent chow 5240 and tap water. Body weight, foodconsumption, and water consumption is recorded daily. Blood samples of0.5 mL are recovered from the catheter once per day during treatment andtwice a week during the three week post infusion interval. Bloodchemistry evaluation and urinalysis are also performed. Blood chemistryanalysis preferably includes LDH, AST, alkaline phosphatase, blood ureanitrogen, plasma creatine concentration, bilirubin, glucose, albumin,and total protein. Blood analysis includes white cell count, red cellcount, hemoglobin, and hematocrit. Radioactivity in blood is determinedby liquid scintillation counting. Gel electrophoresis is preferablyperformed to determine the size distribution of the radioactivematerial. Blood chemistry is evaluated on days -1, 1, 3, 5, and 7 of theinfusion and once per week during the three week post-infusion interval.The total urine volume is also measured daily. Analysis of urine forcolor, pH, radioactivity, and dipstick is preferably performed on each24 hour urine sample. Gel electrophoresis is performed on samples todetermine the size distribution of the radioactive material in theurine. If significant toxicity is observed at this dose an additionalrepeat of this study may be performed at 300 mg/m². If no toxicity isobserved then a safe margin is indicated.

B. Methods for Determining the Biological Activity of FdU_(n) and

FrU_(n) Compounds Against Tumors in a Mouse Model

The potential clinical utility of a few of the oligomeric5-fluorouridine compounds most promising in the cytotoxicity assaysdescribed before are preferably assessed via a mouse melanoma model. TheB16-BL6 melanoma is available from the DCT Tumor Bank, National CancerInstitute, Frederick, Md. This is a highly invasive and metastaticsubline of the B16 melanoma originally isolated by Fidlet. The tumor ispreferably maintained in plastic tissue culture flasks in a humidifiedatmosphere containing 5% CO₂ in air. The culture medium used is minimalessential medium supplemented with 10% fetal calf serum, 2 mM glutamine,0.1 mM non-essential amino acids, vitamins, a mixture of 100 U/mLpenicillin and 100 μg/mL streptomycin and designated complete essentialmedium (CMEM). Each C57BL/6J mouse is given a subcutaneous injection of5×10⁴ viable tumor cells (0.1 mL) into the right flank. The subcutaneoustumors are measured with a vernier caliper. Measurements are calculatedpreferably as the geometric mean of two diameters, the first being thelongest diameter and the second being measured perpendicular to thefirst diameter. Tumor growth is followed for 21 days. Four groups of 10mice (5 male/5 female) are used for each compound. The first groupreceives saline solution while groups 2, 3, and 4 receive 50, 150, and300 mg/m² of oligomeric 5-fluorouridine compounds delivered daily forseven days following the day after tumor inoculation. The oligomeric5-fluorouridine compound is infused by using 14 day Alzet infusion pumpsimplanted subcutaneously. Mice are fed and cared for as in the rattoxicity study described before. Control experiments with 5-fluoruracilare also conducted simultaneously. Efficacy of the oligomeric5-fluorouridine compounds are measured in their absolute success ininhibiting tumor growth and in their success relative to 5-FU atequivalent and lower doses.

The oligomeric 5-fluorouridine compounds tested against the mousemelanoma model are also preferably tested in mice against Lewis lungcarcinoma. The Lewis lung carcinoma was obtained from the Division ofCancer Treatment Tumor Bank (National Cancer Institute), FrederickCancer Research Facility (Frederick, Md.). The tumor is cultured inplastic tissue culture flasks in a humidified atmosphere of 5% CO₂ inair. The Lewis lung carcinoma cells are cultured on media consisting ofDulbecco's minimal essential medium and glutamine, supplemented with 10%fetal calf serum, 100 IU penicillin/mL, and 100 ug streptomycin/mL.Cells are harvested after treatment of the monolayers with a mixture oftrypsin (0.5%) and EDTA (0.2%) for one minute. The single cells arewashed in media and resuspended in phosphate buffered saline (PBS).Viable mammary carcinoma cells (5×104 in 0.2 mL of PBS) are injectedinto the tail vein of C57BL/6J mice. The same feeding, care, and drugadministration protocols described for the mouse melanoma model arefollowed. The rate and growth of the primary tumors is measured withcalipers. In addition, after three weeks the animals are sacrificed andthe lungs are removed and fixed in buffered formalin for 24 h and thenumber of metastatic foci on the surface of the lung are counted withthe aid of a dissecting microscope.

V. Utilization of FdU_(n) and FrU_(n) as a Chemotherapeutic Agents

Homo-oligomeric FdUMP and homo-oligomeric FUMP or their conjugates areto be used clinically as anti-cancer agents. Tumor targets are thesolid-tumors of the breast, head, neck, colon and elsewhere that arecurrently targets for 5-FU chemotherapy. An intravenous blousadministration is currently thought preferred (5-fluorouraciladministration approved protocols are followed). Combinationchemotherapy involving leucovorin and B-interferon is also expected tobe effective with these homopolymers as for the nucleoside andnucleoside base. It is anticipated that therapeutically active doses ofFdU_(n) and FrU_(n) will deliver less 5-fluoruracil because of their (1)greater bioavailability (due to longer residence time in-vivo); (2)efficiency of cellular uptake; and (3) natural degradation directly tothe active metabolites. The lower doses (i.e. less 5-fluorouracil)produce lower levels of FBAL and are less neurotoxic and cardiotoxic,thus improving the therapeutic index of the drug relative to delivery as5-fluorouracil.

It should be apparent to those skilled in the art that anon-homooligomer containing antimetabolites such as 5-FU interspersed ina sequence of other nucleotides or nucleotide analogues will providesimilar advantages to the homo-oligomer. This would includeoligonucleotides with sequences capable of modulating gene expressionthrough antisense, antigen, or as binding sites for endogenous materialswhich are involved in the process of transcription. Finally,preparations of oligonucleotides capable of forming double, triple orquartet strands represent an extension of the fundamental advantages ofa homo-oligomer.

VI. Conclusion

Thus, there has been described: (1) a method of synthesizing usefuloligomeric species of 5-fluorouracil; (2) methods of utilizingoligomeric species of 5-fluorouracil in the treatment of solid tumorswherein bioavailibility is increased; (3) and methods of enhancing thedelivery of oligomeric species of 5-fluorouracil such that (a) cytotoxicmetabolite delivery is timed for optimum tumor cell lethality, (b) thecytoxic metabolites are tumor target specific, and (c) tumor cell uptakeis optimized.

It will be apparent to those both skilled in the art, and familiar withthe present disclosure, that many modifications and additions may bemade to the present invention, for example, (1 ) other methods ofsynthesis may be utilized and the principles disclosed herein may beadapted for preparing oligomers of other chemotherapeutic agents; (2)other methods of utilizing oligomeric species of 5-fluorouracil for thetreatment of diseases are anticipated wherein the general principles ofthe instant invention are utilized; (3) and other methods of enhancingthe delivery of oligomeric compounds are also anticipated such that (a)drug delivery is timed for optimum pharmacokinetic effect, (b) targetspecific drugs are produced, and (c) adverse effects are minimized.

Thus, there has been described a method of increasing the efficacy oftreatments utilizing 5-fluorouracil which accomplishes at least all ofthe stated objects, including disclosure teaching the modification ofother bio-active nucleoside analogues.

We claim:
 1. A homo-oligonucleotide consisting essentially of between 2and 26 monomers of 5-fluorodeoxyuridine 5'-monophosphate (FdUMP)covalently linked via 3'- to 5'-phosphodiester linkages, where at the3'- or 5'-terminus there is covalently linked a molecule selected fromthe group consisting of cholesterol, ethyl-spaced adamatane,1,2-di-hexadecylglycerol and poly-L-lysine.
 2. The homo-oligonucleotideof claim 1 wherein said molecule is covalently linked only at the3'-terminus.
 3. The homo-oligonucleotide of claim 1 wherein saidmolecule is covalently linked only at the 5'-terminus.
 4. Ahomo-oligonucleotide consisting essentially of between 2 and 26 monomersof 5-fluorouridine 5'-monophosphate (FUMP) covalently linked via 3'- to5'-phosphodiester linkages, where at the 3'- or 5'-terminus there iscovalently linked a molecule selected from the group consisting ofcholesterol, ethyl-spaced adamatane, 1,2-dihexadecylglycerol andpoly-L-lysine.
 5. The homo-oligonucleotide of claim 4 wherein saidmolecule is covalently linked only at the 3'-terminus.
 6. Thehomo-oligonucleotide of claim 4 wherein said molecule is covalentlylinked only at the 5'-terminus.
 7. A homo-oligonucleotide consistingessentially of between 2 and 26 monomers of 5-fluorodeoxyuridine5'-monophosphate (FdUMP) covalently linked via 3'- to 5'-phosphothioatelinkages, where at the 3'- or 5'-terminus there is covalently linked amolecule selected from the group consisting of cholesterol, ethyl-spacedadamatane, 1,2-di-hexadecylglycerol and poly-L-lysine.
 8. Thehomo-oligonucleotide of claim 7 wherein said molecule is covalentlylinked only at the 3'-terminus.
 9. The homo-oligonucleotide of claim 7wherein said molecule is covalently linked only at the 5'-terminus. 10.A homo-oligonucleotide consisting essentially of between 2 and 26monomers of 5-fluorouridine 5'-monophosphate (FUMP) covalently linkedvia 3'- to 5'-phosphothioate linkages, where at the 3'- or 5'-terminusthere is covalently linked a molecule selected from the group consistingof cholesterol, ethyl-spaced adamatane, 1,2-dihexadecylglycerol andpoly-L-lysine.
 11. The homo-oligonucleotide of claim 10 wherein saidmolecule is covalently linked only at the 3'-terminus.
 12. Thehomo-oligonucleotide of claim 10 wherein said molecule is covalentlylinked only at the 5'-terminus.